涡鞭毛虫（甲藻）着丝粒／动粒蛋白的检查

利用ＡＣＡ血清、抗人着丝粒蛋白Ｂ的单抗和多抗、抗ＣＨＯ细胞动粒蛋白的单抗,对典型涡鞭毛虫隐沟虫（隐甲藻）（Ｃｒｙｐｔｈｅｃｏｄｉｎｉｕｍｃｏｈｎｉｉ）和特殊涡鞭毛虫尖尾虫（尖尾藻）（Ｏｘｙｒｒｈｉｓｍａｒｉｎａ）的着丝粒／动粒蛋白进行了检查。用ＡＣＡ血清作的荧光观察表明,隐沟虫的这些蛋白虽结合在核骨架上,但在间期时并不形成点状的前着丝粒。免疫印迹检查表明两种涡鞭毛虫的着丝粒蛋白Ｂ彼此一致,而且与四膜虫和眼虫的也高度一致。但用ＡＣＡ血清作免疫印迹检查时,尖尾虫的蛋白虽与四膜虫和眼虫的相近,与隐沟虫的却有极大的差异。以抗动粒蛋白的单抗作此种检查时,尖尾虫与眼虫的反应带相同,而隐沟虫则与源真核生物（Ａｒｃｈｅｚｏａ）贾第虫（Ｇｉａｒｄｉａｌａｍｂｌｉａ）的相同;而且隐沟虫和贾第虫都与几种原细菌有两条相同的反应带,其中５０ｋＤ的一条是尖尾虫和眼虫都没有的。上述发现不仅从一个新的方面支持了认为应把尖尾虫从典型涡鞭毛虫分出来独立为一个门的主张（李靖炎,１９９０）,而且指出典型涡鞭毛虫在后真核生物（Ｍｅｔａｋａｒｙｏｔａ）中间是非常原始的。     

小眼虫的着丝粒蛋白检查

为了从起源与进化的角度考察丝粒蛋白,我们从比酵母更低等的原生生物着手,检查它们的着丝粒蛋白,另文我们已报道了对四膜虫研究的结果,本文报道的是对小眼虫（Ｅｎｇｌｅｎａｇｒａｃｉｌｌｓ）的检查。我们的结果表明：眼虫细胞核里存在的ＡＣＡ抗原的分子量,包括了用ＡＣＡ血清在高等生物中检出来的ＣＥＮＰ－Ａ,ＣＥＮＰ－Ｂ,ＣＥＮＰ－Ｃ和ＣＥＮＰ－Ｄ这几类基本的着丝粒蛋白组分,作免疫荧光染色后,细胞核呈很强的阳性     

源真核生物蓝氏贾第虫核分裂的初步观察

贾第虫属于源真核生物（Ａｒｃｈｅｚｏａ）中的双滴虫门,是目前所知的最低等的真核生物。本工作首次对蓝氏贾第虫（Ｇｉａｒｄｌｉａｌａｍｂｌａ）的核分裂作了初步的电镜观察,未能在分裂着的核中见到纺锤体或纺锤体微管。以０．１—２０μｇ／ｍｌ浓度的秋水仙素作实验,其核分裂也不受阻抑。以抗微管蛋白的多抗作免疫荧光检查,也未见分裂着的核中有微管蛋白。这似乎意味着其核分裂方式乃是前有丝分裂性质的。对此进行了讨论。     

蓝氏贾第虫核纤层蛋白基因的初步研究

贾第虫一度被认为是迄今已知的最原始的真核细胞,但近来争议日盛。利用PCR和测序等技术,对蓝氏贾第虫(Giardia lamblia)的核纤层蛋白(lamin)基因进行了研究。结果表明：蓝氏贾第虫基因组中存在一个编码具有明显lamin特征的基因序列。如该基因序列的3’一端具有编码与核内膜亲和的特征性模体(motif)CaaX的序列;具有B型lamin基因所特有的高度保守的27bp片段,该片段编码高度保守的位于a螺旋杆状区的9氨基酸片段等。同时,这些序列特征又与多细胞的后生动物存在一定差异。这些事实说明在贾第虫中已经进化产生了典型真核细胞的B型lamin(基因)或至少是类似B型的lamin(基因),该生物的进化地位可能并非过去所认为的那么原始。     

蓝氏贾第虫无义mRNA的降解

无义介导的mRNA降解途径（nonsense-mediated mRNA decay,NMD）作为细胞内的一种重要的mRNA质量监控机制,可以降解含有提前终止密码子（premature termination codon,PTC）的异常转录本,从而避免截短蛋白质对细胞的毒害,但其详细的分子机制有待进一步阐释。蓝氏贾第虫（Giardia lamblia)作为一种寄生性单细胞原生动物,进化地位特殊,对其NMD途径的研究有利于阐明基因表达调控的分子和进化机制。本研究通过酵母双杂交及体外pull-down实验分析了贾第虫NMD途径因子上游移码蛋白1(Giardia lamblia up-frameshift 1,GlUPF1)、贾第虫RNA结合蛋白（Giardia lamblia HRP1, GlHRP1）、贾第虫核糖核酸外切酶（Giardia lamblia Ski7p,GlSki7p、Giardia lamblia XRN1,GlXRN1）之间的相互作用关系。结果表明,GlUPF1全长与GlHRP1、GlXRN1(1~500 aa)、GlSki7p间均可发生相互作用。而且GlUPF1的CH结构域和C端结构域分别与GlHRP1、GlXRN1（1~500 aa）、GlSki7p相互作用。说明GlUPF1在贾第虫NMD途径中作为招募平台,在无义mRNA识别和降解过程中发挥重要作用。为此,结合本实验室之前的研究结果,我们提出原生动物贾第虫的NMD途径：在提前终止密码子处SURF(SMG1-UPF1-eRF1-eRF3)复合物形成后,GlUPF1被磷脂酰肌醇3-激酶(suppressor with morphogenetic effect on genitalia 1,SMG1)磷酸化修饰, NMD途径激活,随后GlUPF1与HRP1相互作用,将转录本标记为NMD底物;GlUPF1进而招募下游贾第虫5′-3′核糖核酸降解酶GlXRN1、贾第虫3′-5′ 核糖核酸降解因子GlSki7p,最终降解靶标mRNA。     

蓝氏贾第虫组蛋白的初步研究

本文初步比较分析了贾第虫组蛋白的存在及其基本组成。通过甲醇固定,两次０．３ｍｏｌ／ＬＨＣｌ抽提及丙酮沉淀,提取贾第虫酸溶性蛋白,利用亲和层析制取总ＤＮＡ结合蛋白,经酸性尿素系统及ＳＤＳ系统电泳分析表明,贾第虫已存在５种组蛋白,其在两种不同性质电泳系统中的电泳行为与相应的小牛胸腺组蛋白有近似的对应关系。说明贾第虫的组蛋白已发生了分化,并已初步形成了性质不同的几个组份,从而在一定程度上支持核小体组蛋白在真核生物的原核祖先阶段就已产生了分化的假说。     

Overlapping genes in parasitic protist Giardia lamblia.

The parasitic protist Giardia lamblia lacks mitochondria and peroxisomes, as well as many typical membrane-bound organella characteristics of higher eukaryotic cells, together with extremely economized usage of DNA sequence, as demonstrated by the lack of introns. We describe here the presence of overlapping genes in G. lamblia, in which a part of the protein coding sequence of one mRNA exists in a region corresponding to the 3′-noncoding region of another mRNA transcribed from a gene on the opposite strand. Recently we isolated 13 kinesin-related cDNAs from G. lamblia. Nine of these cDNAs contain long 3′-noncoding sequences in which long open reading frames (ORFs) exist (in the remaining four cDNAs, the lengths of the 3′-noncoding sequences are very short). The predicted amino acid sequences of these ORFs were subjected to a search for homologies with sequences in databases. The amino acid sequences of the six ORFs exhibited significant sequence similarities with known sequences. These lines of evidence suggest the frequent occurrence of gene overlap in Giardial genome.     

Expression of tubulin polyglycylation in Giardia lamblia

By means of immunofluorescence, immunoelectron microscopy and immunoblotting, we show that polyglycylation, a posttranslational modification of tubulin widely spread among eukaryotes, is present in the diplomonad, Giardia lamblia, a putative ancestral cell possessing a highly developed microtubular cytoskeleton. This modification was recently discovered in the ciliated protist, Paramecium, and was not found in the Euglenozoa, a lineage considered as ancient. We used two monoclonal antibodies (mAbs), TAP 952 and AXO 49, specifically recognizing mono- and polyglycylated tubulin isoforms, to detect this modification in Giardia extracts and to localize it in the different classes of microtubules within the cell. The alpha- and beta-tubulin subunits were recognized by the two mAbs, indicating that both tubulin subunits are glycylated, in agreement with lately reported mass spectrometry results. Noticeably, Giardia tubulin was much more reactive with AXO 49 than with TAP 952. In situ, AXO 49 intensely labeled the microtubules present in the four pairs of flagella and the median body, and lightly decorated the microtubules from the adhesive disc. In contrast, TAP 952 intensely labeled only the microtubules of the median body. The results indicate a differential expression of glycylated isoforms within various microtubular structures of Giardia lamblia. They also suggest that the complete set of enzymes required for polyglycylation is expressed in very divergent eukaryotes.     

Solution-phase parallel synthesis of substituted chalcones and their antiparasitary activity against Giardia lamblia

A library of 25-membered chalcones was prepared by parallel synthesis. Substituted acetophenones and benzaldehydes were condensed using the Claisen–Schmidt base-catalyzed aldol condensation. Several chalcones showed in vitro antiparasitic activity against Giardia lamblia. The highest activity observed for the IC₅₀ values were 12.72, 15.05 and 15.31 μg/mL, respectively; these are potential leads for the development of antigiardial compounds.     

原细菌是真核细胞的祖先吗?

真核细胞是由什么样的原核细胞进化而来？这是一个长期困扰人们的基本生物学问题。但近年来随着原细菌的发现和广泛深入的研究,使这一问题的解决向前迈进了一大步。结合国际上对原细菌,尤其是它与真核细胞在许多方面（包括ＤＮＡ复制、转录和翻译整个中心法则,组蛋白、染色质及蛋白酶复合体等）密切的亲缘关系的研究结果,对原细菌的进化地位及其对揭示真核细胞的起源进化问题的意义进行了探讨。     

从基因组分析贾第虫的核糖体合成系统

为探讨贾第虫细胞核内核糖体合成系统,及与典型的真核生物有何差异,首先,确定在典型真核生物中参与核糖体合成的129条共有的保守蛋白,然后用这些蛋白搜索贾第虫基因组以调查它们在贾第虫中的直系同源蛋白的情况,以对贾第虫的核糖体合成系统作一了解。结果表明：贾第虫具有89条这些蛋白的直系同源蛋白,包括参与rRNA甲基化和假尿嘧啶化的蛋白复合体成员,以及存在于90S、40S和60S复合体中的蛋白。贾第虫的核糖体合成系统与典型的真核生物相似,但还有40条蛋白在贾第虫基因组中找不到同源蛋白。这意味着贾第虫的核糖体合成系统较典型的真核生物简单。贾第虫虽然没有核仁结构,但其核糖体亚基合成的途径和机制可能与真核细胞相似,参与的成分不同于无核仁结构的原核生物,可能相对简单。